α1‐Proteinase inhibitor in gingival crevicular fluid of humans with adult periodontitis: serpinolytic inhibition by doxycycline

The serum protein, alpha 1-proteinase inhibitor (alpha 1-PI), defends the host against serine proteinases, e.g. PMN elastase. Using a rabbit anti-serum against human alpha 1-PI, this protein in GCF was quantified from a standard curve constructed from dot-blot analysis and characterized by Western blot. GCF was collected on filter paper strips from healthy (H), gingivitis (G) and adult periodontitis (AP) patients, then extracted with Tris/NaCl/CaCl2 buffer, pH 7.6. alpha 1-PI concentration increased with G and was highest in AP subjects. H sites only showed intact alpha 1-PI (52 kDa); no degradation fragments (48 kDa) were detected. In G and AP subjects, alpha 1-PI degradation fragments were seen in 17% and 71% of GCF samples, respectively. Both collagenase and alpha 1-PI-degrading activities in GCF increased with severity of inflammation (GCF flow). Moreover, the alpha 1-PI degrading (or serpinolytic) activity was characterized as a matrix metalloproteinase, probably collagenase, based on its in vitro response to a panel of different proteinase inhibitors including doxycycline. We propose: (1) that collagenase promotes periodontal breakdown not only by degrading collagen, but also by depleting alpha 1-PI regulation of elastase and other serine-proteinases, thereby favoring a broader attack on extracellular matrix (ECM) constituents, and (2) based on a recent longitudinal double-blind study using the techniques described above for alpha 1-PI analysis, that low-dose doxycycline administration to humans with adult periodontitis can inhibit this broad cascade of ECM degradation.